Advanced NIR ratiometric probes for intravital biomedical imaging

Near-infrared fluorescence imaging technology (NIR-I region, 650–950 nm and NIR-II region, 1000–1700 nm), with deeper tissue penetration and less disturbance from auto-fluorescence than that in visible region (400−650 nm), is playing a more and more extensive role in the field of biomedical imaging. With the development of precise medicine, intelligent NIR fluorescent probes have been meticulously designed to provide more sensitive, specific and accurate feedback on detection. Especially, recently developed ratiometric fluorescence probes have been devoted to quantify physiological and pathological parameters with a combination of responsive fluorescence changes and self-calibration. Herein, we systemically introduced the construction strategies of NIR ratiometric fluorescence probes and their applications in biological imaging in vivo, such as molecular detection, pH and temperature measurement, drug delivery monitoring and treatment evaluation. We further summarized possible optimization on the design of ratiometric probes for quantitative analysis with NIR fluorescence, and prospected the broader optical applications of ratiometric probes in life science and clinical translation.

Multi-Photon Microscopy for the Evaluation of Interstitial Fibrosis in Extended Criteria Donor Kidneys: A Proof-of-Concept Study

Introduction:This study evaluated the initial use of label-free second harmonic generation (SHG) imaging with two-photon excitation (2PE) auto-fluorescence for the quantification of collagen/fibrosis on pre-implantation biopsies of extended criteria donors (ECD).Methods:A total of 20 core ECD kidney transplant biopsy specimen tissues from 7 ECD donors, taken at the time of pre-implantation were retrieved, cut into 5-micron sections, mounted on slides and deparaffinized. The core needle biopsies were imaged with 2X and 20X objective using the commercially available laser-based Genesis® 200 (Histoindex Pte Ltd and Clinnovate Pte Ltd, Singapore). The entire core was selected as the Region of Interest (ROI). Corresponding clinical information from transplant donors were retrieved. Histopathological review was performed, and all biopsies had Interstitial Fibrosis (IF)/Tubular Atrophy (TA) scores > 0. Collagen parameters measured included quantification by the Collagen Area Ratio in Tissue (CART) and qualitative measurements by Collagen Reticulation Index (CRI).Results: 20 explant core biopsies were extracted from 10 donor kidney samples, of which originated from 7 donors. Table 1 depicts the baseline ECD characteristics of the donors. For the kidneys with multiple biopsies done, we obtained an average score of the collagen parameters across the different samples. Biopsies classified with > 85% KDPI score had significantly higher CAR and CART than biopsies with ≤ 85% KDPI score.Conclusion:MPM is an evolving technology that enables the quantification of the amount (CART) and quality (CRI) of collagen deposition in unstained explant biopsies of ECD kidneys. This initial evaluation found significant differences in both parameters between ECD kidneys with more or less than 85% KDPI scores.

Multi-Photon Microscopy for the Evaluation of Interstitial Fibrosis in Extended Criteria Donor Kidneys: A Proof-of-Concept Study

Introduction:This study evaluated the initial use of label-free second harmonic generation (SHG) imaging with two-photon excitation (2PE) auto-fluorescence for the quantification of collagen/fibrosis on pre-implantation biopsies of extended criteria donors (ECD).Methods:A total of 22 core ECD kidney transplant biopsy specimen tissues from 7 ECD donors, taken at the time of pre-implantation were retrieved, cut into 5-micron sections, mounted on slides and deparaffinized. The core needle biopsies were imaged with 2X and 20X objective using the commercially available laser-based Genesis® 200 (Histoindex Pte Ltd and Clinnovate Pte Ltd, Singapore). The entire core was selected as the Region of Interest (ROI). Corresponding clinical information from transplant donors were retrieved. Histopathological review was performed, and all biopsies had Interstitial Fibrosis (IF)/Tubular Atrophy (TA) scores > 0. Collagen parameters measured included quantification by the Collagen Area Ratio in Tissue (CART) and qualitative measurements by Collagen Reticulation Index (CRI).Results: 20 explant core biopsies were extracted from 10 donor kidney samples, of which originated from 7 donors. Table 1 depicts the baseline ECD characteristics of the donors. For the kidneys with multiple biopsies done, we obtained an average score of the collagen parameters across the different samples. Biopsies classified with > 85% KDPI score had significantly higher CAR and CART than biopsies with ≤ 85% KDPI score.Conclusion:MPM is an evolving technology that enables the quantification of the amount (CART) and quality (CRI) of collagen deposition in unstained explant biopsies of ECD kidneys. This initial evaluation found significant differences in both parameters between ECD kidneys with more or less than 85% KDPI scores.

Submicron Non-contact Simultaneous Infrared and Raman Spectroscopy for Challenging Failure Analysis

We introduce a new infrared (IR) technique that provides submicron spatial resolution by making use of an infraredvisible, pump-probe arrangement that also offers a simultaneous Raman measurement in formerly challenging failure and contamination analyses. These challenges are typically due to the lack of spatial resolution and sample preparation restrictions from conventional FTIR, plus auto-fluorescence (AF) from Raman spectroscopy. Such a combined Optical PhotoThermal InfraRed (O-PTIR) and Raman instrumentation offers spatial resolution improvement over conventional IR measurements by 30 times at 1000 cm-1. The technique also improves sensitivity to exceptionally small quantities (? 400 femtogram) in reflection mode by sensing the photothermal response arising from absorbing infrared radiation (Fig. 1) [1]. The AF-free O-PTIR technique also delivers constant spatial resolution over the entire mid-IR range due to the use of a fixed wavelength probe beam at 532 nm [2]. Simultaneous Raman confirms and complements the O-PTIR measurements in cases with low AF. We will illustrate three examples that will highlight the advantage of the novel technique commonly observed in the failure and contamination analysis community.

Microstructural Analysis of Whey/Soy Protein Isolate Mixed Gels Using Confocal Raman Microscopy

This work explores the potential of confocal Raman microscopy to investigate the microstructure of mixed protein gel systems. Heat-set protein gels were prepared using whey protein isolate (WPI), soy protein isolate (SPI), and mixtures thereof, with a total of five different whey-to-soy protein ratios (100, 75, 50, 25, and 0%). These were analysed using confocal Raman microscopy, and different data analysis approaches were used to maximize the amount of structural and compositional information extracted from the spectral datasets generated, including both univariate and multivariate analysis methods. Small spectral differences were found between pure WPI and SPI gels, mainly attributed to conformational differences (amide bands), but SPI exhibited considerably greater auto-fluorescence than WPI. The univariate analysis method allowed for a rapid microstructural analysis, successfully mapping the distribution of protein and water in the gels. The greater fluorescence of the capsule-like structures found in the mixed gels, compared to other regions rich in proteins, suggested that these may be enriched in soy proteins. Further analysis, using a multivariate approach, allowed us to distinguish proteins with different levels of hydration within the gels and to detect non-proteinaceous compounds. Raman microscopy proved to be particularly useful to detect the presence of residual lipids in protein gels.

Auto Fluorescence Allows Us to Detect Early Signs of Oral Cancer and Much More

This paper introduces a novel approach, using autofluorescence, to objectively examine the oral cavity for inflammation and infection. Many systemic diseases are perpetuated by microorganisms that colonize in the oral environment. They enter the cardiovascular system by enzymatic processes that open the oral mucosa to allow their entry. A majority of the microbes are anaerobes and/or facultative anaerobes. When they enter the host, they metabolize blood. Their waste by products contains iron elements within a compound called porphyrin. Porphyrin will fluoresce when exposed to certain wavelengths of light. Healthcare providers can utilize this natural occurring process to objectively see these harmful pathogens. This may indicate that the host has a Sleep Related Breathing Disorder (SRBD). Sleep apnea is a primary disorder of SRBD’s. This technology offers medical and dental fields a screening tool for a pandemic healthcare problem.

Multi-Spectral Clinical Prototype for Fluorophore Detection

Identification of tumour margins during resection of the brain is critical for improving the post-operative outcomes. Current methods of tumour identification use 5-ALA, an exogenous precursor, metabolized to fluorescent PpIX in tumour tissue. Although visible under fluorescent microscope, PpIX is easily photo-bleached and tumour tagging is subjective, resulting in tumour under-resection and accelerated recurrence. To address this issue, photo-bleaching resistant and quantitative method is required. This study describes the characterization of a pulsed, multi-wavelengths system designed to measure diffuse reflectance and auto-fluorescence under strong ambient illumination conditions. The performance was tested on n = 400 liquid tissue phantoms containing a wide concentration range of absorber, scatterer and two fluorophores as well as on ex-vivo samples of gray and white matter. The background subtraction technique was shown to be efficient for a range of ambient illumination intensities. A linear relationship was observed between system response and predicted fluorophore concentrations as well as 97.8% accuracy of tissue classification by 5-fold cross-correlation, linear SVM.

Fluorescence-guided Surgery of Osteoradionecrosis of the Jaw: A Retrospective Study

BackgroundOsteoradionecrosis of the jaw (ORNJ) is one of the most severe head and neck complications in patients treated with radiotherapy (RT), and it is one of the diseases difficult to manage. The goal of the treatment is to achieve mucosal healing or suppress ORNJ progression. Currently, surgical removal of the necrotic bone is an effective management approach for advanced stages of ORNJ. This study aimed to identify the outcomes of fluorescence-guided surgery for ORNJ. MethodsNineteen ORNJ lesions in 15 hospitalized patients received fluorescence-guided surgery. Demographics, comorbidities, local preceding event, location, ORNJ stage, and treatment outcomes were retrospectively reviewed with a median follow-up period of 12 months.ResultsThe first 12 lesions (63%) were operated under tetracycline fluorescence, and 7 lesions (37%) were operated under auto-fluorescence. Overall, 4 lesions (21%) achieved complete mucosal healing, 8 lesions (42%) showed partial mucosal healing with bone exposure and no signs or symptoms of inflammation, and 7 lesions (37%) were progressive. The results showed that either healing or ORNJ stabilization was achieved in 63% of the lesions (n = 12). No significant association was observed between healing and the fluorescence technique.ConclusionFluorescence-guided surgery can be beneficial in curing or stabilizing ORNJ. Within the study limitations, autofluorescence-guided surgery seems to be as effective as tetracycline fluorescence-guided surgery for management of ORNJ. Trial registration: not applicable

Evaluation of Neuraminidase Inhibitory Activity of Compounds and Extracts from Traditional Medicines by HPLC-FLD

A simple and effective method was established and validated to determine 4-methylumbelliferone (4-MU) for screening the natural neuraminidase inhibitors (NAIs) from traditional medicines (TMs) by high performance liquid chromatography combined with fluorescence detection (HPLC-FLD). 4-MU and TMs compounds were separated on a Hedera TM ODS column (5 μm, 4.6 × 250 mm) using an isocratic elution of 55% methanol at 35°C. The flow rate was 1 mL min−1. The excitation and emission wavelength were performed at 320 nm and 480 nm. Some extracts of TMs and compounds were selected as examples to demonstrate the feasibility of the new HPLC-FLD method. It was found that the results of most compounds except for the auto fluorescence substances determined by HPLC-FLD were in good agreement with NA enzyme-based inhibitory assays. Comparing to traditional NA enzyme-based inhibitory assays, the HPLC-FLD method could prevent interference from fluorescence pigments of compounds. It was considered a simple, effective, and economical technique for the screening the natural neuraminidase inhibitors from traditional medicines.

A method for simultaneously monitoring phloem and xylem reconnections in grafted watermelon seedlings

Grafting is an effective way to increase watermelon tolerance to biotic and abiotic stresses. However, the survival of grafted seedlings largely depends on successful graft formation. Therefore, understanding the graft formation process, particularly the vascular reconnection process is of critical importance. This study found that lignin in watermelon stem shows strong auto-fluorescence under blue-light excitation which makes blue-light excited fluorescent tracers (FTs) such as 5(6)-carboxy fluorescein diacetate (CFDA) become unsuitable for assaying vascular connectivity in watermelon. In contrast, UV-light excited esculin and red-light excited acid fuchsin were proved to be efficient FTs for monitoring the phloem and xylem connectivity, respectively, in self-grafted watermelon. Furthermore, a combined application of esculin to the scion cotyledon and acid fuchsin to the rootstock root enabled simultaneous monitoring of the phloem and xylem connectivity in individual self-grafted watermelon seedlings. In addition, this method is also applicable in investigating the phloem and xylem reconnections in self-grafted melon and cucumber, and heterograft of watermelon, melon and cucumber onto pumpkin rootstock. Based on this established method, we found that phloem and xylem reconnections are not timely separated in self-grafted watermelon. Furthermore, low temperature and removal of the rootstock cotyledons both delayed the vascular reconnection process in watermelon. In conclusion, this new method provides a convenient, accurate and rapid way to analyze the vascular connectivity not only in watermelon, but also in other cucurbit crops.